Persistent, Asymptomatic Colonization with Candida is Associated with Elevated Frequencies of Highly Activated Cervical Th17-Like Cells and Related Cytokines in the Reproductive Tract of South African Adolescents

ABSTRACT Cervicovaginal inflammation, nonoptimal microbiota, T-cell activation, and hormonal contraceptives may increase HIV risk, yet associations between these factors and subclinical Candida colonization or hyphae are unknown. We collected cervicovaginal samples from 94 South African adolescents, aged 15 to 19 years, who were randomized to injectable norethisterone enanthate (Net-En), an etonorgesterol/ethinyl estradiol vaginal ring (NuvaRing), or oral contraceptives in the UChoose trial (NCT02404038) at baseline and 16 weeks post-randomization. We assessed cervicovaginal samples for subclinical Candida colonization (by quantitative PCR [qPCR]), hyphae (by Gram stain), microbiota composition (by 16S rRNA gene sequencing), cytokine concentrations (by Luminex), and cervical T-cell phenotypes and activation (by multiparameter flow cytometry). While hormonal contraceptive type did not influence incidence of Candida colonization or hyphae, hyphae presence was associated with significantly elevated concentrations of IL-22, IL-17A and IL-17F, all produced by Th17 cells, but not of other cytokines, such as IL-1β or IL-6, after adjustment for confounders. Subclinical Candida colonization was associated with reduced frequencies of Th17-like cells and elevated frequencies of CCR6-CCR10 T cells. Women with Candida hyphae were less likely to have bacterial vaginosis (BV). Persistent, subclinical colonization with Candida over 16 weeks was associated with significant increases in Th17-related cytokine concentrations and highly activated Th17-like and CCR6-CCR10 T-cell frequencies. These data suggest that vaginal Candida colonization and hyphae increase Th17-related cytokines, but not overall female genital tract inflammation in Sub-Saharan African adolescents. Persistent Candida colonization, even when asymptomatic, may increase Th17 cell frequencies and related cytokines and thereby could subsequently increase HIV risk, although the causal relationship requires confirmation. IMPORTANCE Sub-Saharan African female adolescents are globally at the highest risk of HIV acquisition, and genital inflammation, microbial dysbiosis, and cervical HIV target cell activation are thought to contribute to this risk. Previously, the relationship between these mucosal factors and subclinical vaginal Candida colonization or hyphae has not been described, and the role of HIV-susceptible Th17 cells in mediating anti-Candida immunity in the human female genital tract has not been clearly established. We show that presence of yeast hyphae was associated with increases in Th17 cell-related cytokines and the absence of microbial dysbiosis, and that persistent Candida colonization resulted in significant increases in Th17-related cytokines and highly activated Th17-like cell frequencies. Our results suggest that Th17 cells are important for anti-Candida immunity in the human female genital tract and that prolonged vaginal Candida colonization may contribute to increased HIV risk in Sub-Saharan African adolescents by increasing HIV target cell frequencies and activation.


General
This was a fascinating study that assesses the relationship between oral contraceptive use, Candida colonization, the greater microbiome, and the immune profile of the female genital tract, all in the context of HIV acquisition risk. These are important questions that have direct clinical applications -e.g., do Candida infections need to be more aggressively treated in those at increased HIV risk. It would be very interesting to explore how symptomatic Candidiasis affects the vaginal immune environment (as no patients reported symptoms in this study), as well as what changes occur with anti-Candida therapies.

Major concerns
The authors use CCR6-,CCR10-cells to indicate Th1/Th2-enriched cells, and CCR6+,CCR10-cells to indicate Th17-enriched cells -is this commonly done? It is concerning that CCR6 can be expressed on Th1 cells as well, and no other makers (other than CD3/CD4) were used. The use of surface markers to designate differentiation is likely not as reliable as using transcription factors or cytokine expression, particularly when used in a relatively limited combination. Perhaps intracellular staining was not possible due to low starting cell number. Regardless, many conclusions are based on this approach, so authors should clarify why these markers were chosen and provide references supporting the use of these phenotyping methods.
The title of the paper asserts that colonization is associated with elevated frequencies of highly activated Th17 cells, but this does not seem to be supported by the data -doesn't Figure 2 show that at baseline, the frequency of Th17 cells overall is decreased in participants with colonization compared to those without? The data do show in figure 6 that percentages of highly activated Th17 cells are increased at 16 weeks in patients with colonization, but it's unclear what the significance of this is, as there is no comparison to those without colonization at 16 weeks.

Minor concerns and remaining questions
In line 163, it is proposed that lower frequencies of Th17-like cells in colonized patients is possibly due to AICD -this should be limited to the discussion section if the authors agree (already appears in lines 265-267), as it is possible but not supported by the data in the results section.
In line 260-261, authors comment that IL-17 and IL-22 are produced by MAIT and Th17 cells, but they are also produced by numerous other cell types -ILCs, NKTs, gamma delta T cells, etc., would be nice to have a mention of this.
In Figure 5A, is the change in hyphae prevalence with Net-En significant?

Technical corrections
Line 86 -missing the word "is" in "there clinical evidence that..." Please ensure abbreviations for full terms are defined on first mention and vice versa. I believe "Net-En" in line 90 is not defined previously (full name appears later in line 92).
Line 210 -typo in the word "the" Figure 1A may be mislabeled -the hyphae column is assigned to the STI legend, and the VVC column to the hyphae legend Reviewer #2 (Comments for the Author): This is an interesting study. In this study, the author described the relationship between asymptomatic vaginal Candida colonization and the frequency of cervical Th17 cells and related cytokines in the reproductive tract of adolescent females in South Africa. Finally, it is concluded that asymptomatic vaginal Candida colonization can increase Th1/Th17 cell frequency and related cytokines. The conclusion of the article has certain significance for reducing the risk of AIDS infection. However, I have the following questions. 1． The number of samples in some groups seems to be too small (<30), which has a certain impact on the accuracy of microbial analysis. 2． Many studies have confirmed that the different physiological stages of women have a great influence on microorganisms. In this study, are all microbial samples collected and unified. 3． I think animal experiments are necessary to explain the relationship between asymptomatic Candida colonization and Th1/Th17 cell frequency. 4． I did not find the address where the 16s sequencing raw data is placed Staff Comments:

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General
This was a fascinating study that assesses the relationship between oral contraceptive use, Candida colonization, the greater microbiome, and the immune profile of the female genital tract, all in the context of HIV acquisition risk. These are important questions that have direct clinical applications -e.g., do Candida infections need to be more aggressively treated in those at increased HIV risk. It would be very interesting to explore how symptomatic Candidiasis affects the vaginal immune environment (as no patients reported symptoms in this study), as well as what changes occur with anti-Candida therapies.

Major concerns
The authors use CCR6-,CCR10-cells to indicate Th1/Th2-enriched cells, and CCR6+,CCR10-cells to indicate Th17-enriched cells -is this commonly done? It is concerning that CCR6 can be expressed on Th1 cells as well, and no other makers (other than CD3/CD4) were used. The use of surface markers to designate differentiation is likely not as reliable as using transcription factors or cytokine expression, particularly when used in a relatively limited combination. Perhaps intracellular staining was not possible due to low starting cell number. Regardless, many conclusions are based on this approach, so authors should clarify why these markers were chosen and provide references supporting the use of these phenotyping methods.
The title of the paper asserts that colonization is associated with elevated frequencies of highly activated Th17 cells, but this does not seem to be supported by the data -doesn't Figure 2 show that at baseline, the frequency of Th17 cells overall is decreased in participants with colonization compared to those without? The data do show in figure 6 that percentages of highly activated Th17 cells are increased at 16 weeks in patients with colonization, but it's unclear what the significance of this is, as there is no comparison to those without colonization at 16 weeks.

Minor concerns and remaining questions
In line 163, it is proposed that lower frequencies of Th17-like cells in colonized patients is possibly due to AICD -this should be limited to the discussion section if the authors agree (already appears in lines 265-267), as it is possible but not supported by the data in the results section.
In line 260-261, authors comment that IL-17 and IL-22 are produced by MAIT and Th17 cells, but they are also produced by numerous other cell types -ILCs, NKTs, gamma delta T cells, etc., would be nice to have a mention of this.
In Figure 5A, is the change in hyphae prevalence with Net-En significant?

Technical corrections
Line 86 -missing the word "is" in "there clinical evidence that…" Please ensure abbreviations for full terms are defined on first mention and vice versa. I believe "Net-En" in line 90 is not defined previously (full name appears later in line 92).
Line 210 -typo in the word "the" Figure 1A may be mislabeled -the hyphae column is assigned to the STI legend, and the VVC column to the hyphae legend Reviewer #1 (Comments for the Author): General This was a fascinating study that assesses the relationship between oral contraceptive use, Candida colonization, the greater microbiome, and the immune profile of the female genital tract, all in the context of HIV acquisition risk. These are important questions that have direct clinical applications -e.g., do Candida infections need to be more aggressively treated in those at increased HIV risk. It would be very interesting to explore how symptomatic Candidiasis affects the vaginal immune environment (as no patients reported symptoms in this study), as well as what changes occur with anti-Candida therapies.

Major concerns
The authors use CCR6-,CCR10-cells to indicate Th1/Th2-enriched cells, and CCR6+,CCR10-cells to indicate Th17-enriched cells -is this commonly done? It is concerning that CCR6 can be expressed on Th1 cells as well, and no other makers (other than CD3/CD4) were used. The use of surface markers to designate differentiation is likely not as reliable as using transcription factors or cytokine expression, particularly when used in a relatively limited combination. Perhaps intracellular staining was not possible due to low starting cell number. Regardless, many conclusions are based on this approach, so authors should clarify why these markers were chosen and provide references supporting the use of these phenotyping methods. We acknowledge that for the conclusive definition of Th1, Th2 or Th17 cells a more complex flow cytometry panel with intracellular staining or evaluation of transcription factors would be required, which we have now mentioned in the discussion. Unfortunately, the number of cervical cells obtainable from cytobrush samples is limited and thus intra-cellular staining after stimulation is not feasible. However, we would like to emphasize that we refer to these cell populations as Th17-enriched or Th17-like, indicating that these cell populations primarily contain Th17 cells but may contain a small proportion of other cell types. For further clarification, we are now referring to CCR6-CCR10-CD4+ T cells as such, rather than referring to this cell population as The title of the paper asserts that colonization is associated with elevated frequencies of highly activated Th17 cells, but this does not seem to be supported by the data -doesn't Figure 2 show that at baseline, the frequency of Th17 cells overall is decreased in participants with colonization compared to those without? The data do show in figure 6 that percentages of highly activated Th17 cells are increased at 16 weeks in patients with colonization, but it's unclear what the significance of this is, as there is no comparison to those without colonization at 16 weeks.
We appreciate the reviewer for pointing this out. We have changed the title to clarify that persistent colonisation over 16 weeks was associated with elevated frequencies of highly activated cervical Th17 cells and related cytokines. To further support this statement, we have amended Figure 6 (and added Figure 7) to include participants who were not colonised over 16 weeks and those acquired or clearing Candida. We further have modified the result and discussion sections of the manuscript to interpret our data more cautiously.

Minor concerns and remaining questions
In line 163, it is proposed that lower frequencies of Th17-like cells in colonized patients is possibly due to AICD -this should be limited to the discussion section if the authors agree (already appears in lines 265-267), as it is possible but not supported by the data in the results section.
We have amended this section of the manuscript and limited these speculations to the discussion.
In line 260-261, authors comment that IL-17 and IL-22 are produced by MAIT and Th17 cells, but they are also produced by numerous other cell types -ILCs, NKTs, gamma delta T cells, etc., would be nice to have a mention of this. We have edited this paragraph to reflect the cell types that contribute to IL-17 and IL-22 production more acutely.
In Figure 5A, is the change in hyphae prevalence with Net-En significant?

Technical corrections
Line 86 -missing the word "is" in "there clinical evidence that..." This has been corrected.
Please ensure abbreviations for full terms are defined on first mention and vice versa. I believe "Net-En" in line 90 is not defined previously (full name appears later in line 92). We have reviewed the manuscript to avoid these errors.
Line 210 -typo in the word "the" This has been corrected. Figure 1A may be mislabeled -the hyphae column is assigned to the STI legend, and the VVC column to the hyphae legend This has been corrected.

Reviewer #2 (Comments for the Author):
This is an interesting study. In this study, the author described the relationship between asymptomatic vaginal Candida colonization and the frequency of cervical Th17 cells and related cytokines in the reproductive tract of adolescent females in South Africa. Finally, it is concluded that asymptomatic vaginal Candida colonization can increase Th1/Th17 cell frequency and related cytokines. The conclusion of the article has certain significance for reducing the risk of AIDS infection. However, I have the following questions.
1． The number of samples in some groups seems to be too small (<30), which has a certain impact on the accuracy of microbial analysis. We agree that the sample size for women with vulvo-vaginal candidiasis, or those acquiring or clearing Candida, is small and thus are limited with regards to the conclusions we can make. The sample size of the substudy was unfortunately limited to that of the parent study, and we have expanded on this limitation in the discussion.
2． Many studies have confirmed that the different physiological stages of women have a great influence on microorganisms. In this study, are all microbial samples collected and unified. Studies have indeed shown that the stage of menstrual cycle may influence vaginal microbiota composition. Since all women were randomised to a certain hormonal contraceptive at baseline, participants were scheduled to return within 40 days of their screening visit for randomisation and samples collected during similar stages of their cycle (at baseline and 16 weeks after hormonal contraceptive initiation). This information has been added to the manuscript.
3． I think animal experiments are necessary to explain the relationship between asymptomatic Candida colonization and Th1/Th17 cell frequency. We agree that animal experiments would be necessary to ultimately determine a causal relationship between Candida colonization and Th1/Th17 cell frequency. This is however beyond the scope of this manuscript. We have emphasized that our findings are based on associations and do not claim causality.
4． I did not find the address where the 16s sequencing raw data is placed Our apologies, this has now been added to the manuscript. You have addressed all reviewers' comments and included a clear section on limitations of the study, including sample size and microbial relationships. Before full acceptance, please perform the edits requested by reviewer 1.
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Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): The reviewers have responded adequately to the previous round of suggestions and comments, and the revised version of the manuscript is clearer and better supported. Only remaining minor suggestion is to edit line 34 of the abstract which currently reads "...significantly elevated concentrations of IL-22, IL-17A and IL-17F, all 34 produced by Th17 cells, but not of pro-inflammatory cytokines"... given that IL-17 can also be pro-inflammatory. Would consider revising more simply to "not of other cytokines such as IL-1 or IL-6." Reviewer #2 (Comments for the Author): For the problem of insufficient sample size, my concerns still cannot be solved. 16s rDNA data are characterized by high sparsity. My concern is whether the various relationships of microorganisms can be well discovered in the case of insufficient sample size.

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Reviewer #1 (Comments for the Author):
The reviewers have responded adequately to the previous round of suggestions and comments, and the revised version of the manuscript is clearer and better supported. Only remaining minor suggestion is to edit line 34 of the abstract which currently reads "...significantly elevated concentrations of IL-22, IL-17A and IL-17F, all 34 produced by Th17 cells, but not of pro-inflammatory cytokines"... given that IL-17 can also be pro-inflammatory. Would consider revising more simply to "not of other cytokines such as IL-1 or IL-6." We have amended this section in the abstract as suggested. It now reads …" hyphae presence was associated with significantly elevated concentrations of IL-22, IL-17A and IL-17F, all produced by Th17 cells, but not of other cytokines, such as IL-1β or IL-6, after adjustment for confounders."